Section III U-Z Vitamin B12 Assay Medium

Vitamin B12 Assay Medium

Intended Use
Vitamin B12 Assay Medium is used for determining vitamin B12 concentration by the microbiological assay technique. is added in specified increasing concentrations providing a growth response that can be measured titrimetrically or turbidimetrically.

Summary and Explanation

Vitamin Assay Media are prepared for use in the microbiological assay of vitamins. Three types of media are used for this purpose: 1. Maintenance Media: For carrying the stock culture to preserve the viability and sensitivity of the test organism for its intended purpose; 2. Inoculum Media: To condition the test culture for immediate use; 3. Assay Media: To permit quantitation of the vitamin under test. They contain all the factors necessary for optimal growth of the test organism except the single essential vitamin to be determined. Vitamin B12 Assay Medium is prepared according to the formula described by Capp, Hobbs and Fox.1 This medium is used in the microbiological assay of vitamin B12 using Lactobacillus delbrueckii subsp. lactis (Lactobacillus leichmannii) ATCC 4797 or 7830.

Formula
Difco Vitamin B12 Assay Medium
Approximate Formula* Per Liter Vitamin Assay Casamino Acids ................................. 12.0 Dextrose ................................................................... 40.0 Sodium Acetate ....................................................... 20.0 L-Cystine .................................................................... 0.2 DL-Tryptophan ........................................................... 0.2 Adenine ................................................................... 20.0 Guanine ................................................................... 20.0 Uracil ........................................................................ 20.0 Xanthine .................................................................... 1.0 Thiamine Hydrochloride ............................................. 2.0 Riboflavin ................................................................... 2.0 Niacin ......................................................................... 2.0 Calcium Pantothenate ............................................ 200.0 Pyridoxine Hydrochloride ............................................ 4.0 p-Aminobenzoic Acid ............................................. 200.0 Biotin ....................................................................... 10.0 Folic Acid ............................................................... 100.0 Polysorbate 80 ........................................................... 2.0 Dipotassium Phosphate .............................................. 1.0 Monopotassium Phosphate ........................................ 1.0 Magnesium Sulfate .................................................... 0.4 Sodium Chloride ...................................................... 20.0 Ferrous Sulfate ......................................................... 20.0 Manganese Sulfate .................................................. 20.0
*Adjusted and/or supplemented as required to meet performance criteria.

Principles of the Procedure

Vitamin B12 Assay Medium is a vitamin B12-free medium containing all other nutrients and vitamins essential for the cultivation of L. delbrueckii subsp. lactis ATCC 4797 or 7830. To obtain a standard curve, USP Cyanocobalamin Reference

g g g g g mg mg mg mg mg mg mg g mg g g g g g g g mg mg mg

Precautions
Great care must be taken to avoid contamination of media or glassware in microbiological assay procedures. Extremely small amounts of foreign material may be sufficient to give erroneous results. Scrupulously clean glassware free from detergents and other chemicals must be used. Glassware must be heated to 250C for at least 1 hour to burn off any organic residues that might be present. Take precautions to keep sterilization and cooling conditions uniform throughout the assay.

User Quality Control

Identity Specifications
Difco Vitamin B12 Assay Medium
Dehydrated Appearance: Solution: Very light to light beige, homogeneous, with a tendency to clump. 3.8% solution (single-strength), soluble in purified water upon boiling 2-3 minutes. Solution is light amber, clear, may have a slight precipitate. Very light amber, clear, may have a very slight precipitate. pH 6.3 0.2

Directions for Preparation from Dehydrated Product

1. Suspend 7.6 g of the powder in 100 mL of purified water. 2. Heat with frequent agitation and boil for 2-3 minutes. 3. Dispense 5 mL amounts into tubes, evenly dispersing the precipitate. 4. Add standard or test samples. 5. Adjust tube volume to 10 mL. 6. Autoclave at 121C for 5 minutes.

Prepared Appearance: Reaction of 3.8% Solution at 25C:

Cultural Response
Difco Vitamin B12 Assay Medium
Prepare the medium per label directions. The medium supports the growth of Lactobacillus delbrueckii subsp. lactis ATCC 4797 when prepared single strength and supplemented with cyanocobalamin (vitamin B12). The medium should produce a standard curve using a cyanocobalamin reference standard at 0.0 to 0.25 ng per 10 mL. Incubate tubes with caps loosened at 35-37C for 18-24 hours. Read the percent transmittance using a spectrophotometer at 660 nm.

Procedure
Stock cultures of the test organism, L. delbrueckii subsp. lactis ATCC 4797 or 7830, are prepared by stab inoculation of Lactobacilli Agar AOAC or B12 Culture Agar. Following incubation at 37C for 24-48 hours, the tubes are stored in the refrigerator. Transfers are made at 2 week intervals.

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Vitamin K1-Hemin Solution

Inoculum for the assay is prepared by subculturing a stock of L. delbrueckii subsp. lactis ATCC 4797 or 7830 into a tube containing 10 mL of Lactobacilli Broth AOAC or B12 Inoculum Broth. After incubation at 35-37C for 18-24 hours, the cells are centrifuged under aseptic conditions and the supernatant liquid decanted. The cells are washed by resuspending in 10 mL of sterile 0.85% saline solution and centrifuging. The washing is repeated for a total of 3 times. Finally the cells are resuspended in 10 mL of sterile 0.85% saline. The cell suspension is then diluted 1:100 with sterile 0.85% saline. One drop is used to inoculate each assay tube. It is essential that a standard curve be constructed each time an assay is run. Conditions of autoclaving and temperature of incubation that influence the standard curve readings cannot always be duplicated. The concentrations required for the preparation of the standard curve are obtained by adding sufficient 25% ethanol to an accurately weighed amount of USP Cyanocobalamin Reference Standard (resulting in a solution containing 1.0 g of cyanocobalamin per mL). This stock solution is stored in the refrigerator and should be used within 60 days. In the preparation of the standard curve, further dilutions of this stock solution (1 g/mL) are made as follows: A. Add 1 mL stock solution to 99 mL purified water (1 mL = 10 ng). B. Add 1 mL of the solution from step A to 199 mL purified water (1 mL = 0.05 ng). An acceptable standard curve can be obtained by using the USP Cyanocobalamin Reference Standard at levels of 0.0, 0.025, 0.05, 0.1, 0.15, 0.2 and 0.25 ng per assay tube. This is accomplished by adding 0, 0.5, 1, 2, 3, 4 and 5 mL of the 0.05 ng/mL solution per assay tube and sufficient purified water to make 10 mL volume per tube. A standard concentration is used which, after incubation, gives a transmittance value at the 5 mL level of not less than that which corresponds to a dry cell weight of 1.25 mg (see USP2 for method of calibration of a spectrophotometer and determination of dry cell weight). For the titrimetric method,

a standard concentration should be used which, after incubation, will give a titration at the 5 mL level of 8-12 mL 0.1N sodium hydroxide. Inoculate and incubate at 35-37C for 18-24 hours. For turbidimetric determinations, place tubes in a refrigerator at 2-8C for 15-20 minutes to stop growth. The growth can be measured by a nephelometric method. Titrimetric determinations of growth are made after incubation at 37C for 72 hours. The curve is then constructed from the values obtained.

Expected Results
1. Prepare a standard concentration response curve by plotting the response readings against the amount of standard in each tube, disk or cup. 2. Determine the amount of vitamin at each level of assay solution by interpolation from the standard curve. 3. Calculate the concentration of vitamin in the sample from the average of these volumes. Use only those values that do not vary more than 10% from the average. Use the results only if two-thirds of the values do not vary more than 10%.

Limitations of the Procedure

1. The test organism used for inoculating an assay medium must be cultured and maintained on media recommended for this purpose. 2. Aseptic technique should be used throughout the assay procedure. 3. The use of altered or deficient media may cause mutants having different nutritional requirements that will not give a satisfactory response. 4. For successful results of these procedures, all conditions of the assay must be followed precisely.

References
1. Capps, Hobbs and Fox. 1949. J. Biol. Chem. 178:517. 2. United States Pharmacopeial Convention, Inc. 2001. The United States pharmacopeia 25/The national formulary 20 2002. United States Pharmacopeial Convention, Inc., Rockville, Md.

U Z

Availability
Difco Vitamin B12 Assay Medium
Cat. No.
*Store at 2-8C

236010

Dehydrated 100 g*

Vitamin K1 - Hemin Solution

Intended Use
Vitamin K1 - Hemin Solution is used as a culture medium enrichment for anaerobic microorganisms.

Principles of the Procedure

Gibbons and MacDonald reported isolating strains of Bacteroides melaninogenicus (Prevotella melaninogenica) that require hemin and vitamin K1 for growth.3 Vitamin K1 enhances the growth of some strains of Bacteroides and certain grampositive nonsporeformers.4 The inclusion of Vitamin K1 - Hemin in anaerobic culture media has been suggested by many investigators.5

Summary and Explanation

CDC Anaerobe 5% Blood Agar was developed at the Centers for Disease Control and Prevention as a nonselective medium for the isolation and cultivation of a wide variety of obligately anaerobic microorganisms, particularly those found in clinical materials.1,2 The medium is enriched with vitamin K1 and hemin.

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